Tandem mass spectrometry method for the genetic screening of inborn errors of metabolism in newborns

ABSTRACT

A method for genetic screening of inborn errors of metabolism in a newborn by tandem mass spectrometry is provided. The method comprises the steps of: (1) placing a blood spot from said newborn into a microtiter plate well; (2) adding a solution of polar solvent to said well so as to reconstitute enzymes in said blood spot and so as to extract polar analytes, thereby forming a sample; (3) incubating said sample for a predetermined period of time, so as to extract metabolites and allow enzyme activity to take place; (4) adding a second solution to said sample so as to stop enzymatic reactions and extract remaining analytes; (5) mixing said sample for a predetermined period of time; (6) measuring said sample by tandem mass spectrometry so as to identify analyte concentration and enzyme activity; and (7) using said analyte concentration and said enzyme activity to determine whether said newborn is affected by any inborn errors of metabolism. The solution of polar solvent may contain at least one of the following: an internal standard for a polar analyte, an internal standard for a non-polar analyte, an internal standard for an enzymatic product, a substrate for at least one enzyme, and a buffer. The second solution may contain at least one of the following: an internal standard for a polar analyte, an internal standard for a non-polar analyte, an internal standard for an enzymatic product, an internal standard for at least one substrate, and a buffer.

TECHNICAL FIELD OF THE INVENTION

The present invention is in the field of spectroscopy. Specifically, thepresent invention is related to methods for use with mass spectrometry.

BACKGROUND OF THE INVENTION

Inborn errors of metabolism (IEM) are genetic disorders in which keyenzymes necessary for the completion of metabolic pathways are inactiveor lacking. Thus affected individuals will either accumulate certainmetabolites to toxic levels and/or lack enzymatic products required forthe completion of essential metabolic pathways. The effects of inbornerrors of metabolism range from mental retardation to prematuredeath.¹⁻³.

As shown in scheme 1, the lack or inactivity of key enzymes due to IEMcan result in the accumulation of precursor metabolites, theaccumulation of secondary product metabolites, and/or the lack ofprimary product metabolites. Because the overriding result of geneticmutations in IEM is the lack or inactivity of essential enzymes, thereare at least two possible ways of detecting these disorders. The firstis to measure the amounts of marker metabolites, i.e. measure theaccumulation of the precursor or alternate product metabolites and/ormeasure the decrease of the primary product metabolite. The secondpossibility is to measure the enzyme activity of the unknown patientsample.

The first approach is generally performed by tandem mass spectrometry(MS/MS) methods. With these MS/MS methods, a small disc from a patientsample in the form of a blood spot dried on filter paper is punched intoa microtiter plate well. A solvent containing a set of internalstandards is added to each well containing a blood spot punch to extractthe marker metabolites from the sample. The solution containing theextracted metabolites and internal standards is then transferred to anew microtiter plate and the solution is dried under heated nitrogen. Tothe dried sample, a new solution containing acid and butanol is added tocreate butyl ester derivatives of the extracted metabolites. Thederivatized sample is then dried under heated nitrogen again to removethe excess derivatizing agent and reconstituted with a third solventtype. Finally, the concentration of the desired metabolites is thenmeasured by isotope dilution MS/MS. This approach is used for measuringthe concentrations of acylcarnitines and amino acids (such as shown inU.S. Pat. Nos. 6,258,605 B1 and 6,455,321 B1 to Chace; herebyincorporated herein by reference). Below is a full protocol for suchMS/MS method for the analysis of amino acids and acylcarnitines:

-   -   1) Prepare extraction stock solution containing internal        standards.    -   2) Make a 1:200 dilution of above (working solution).    -   3) Punch plate and extract with 100 μL of working solution for        30 minutes.    -   4) Transfer extract to a heat resistant plate.    -   5) Dry extract under heated air or nitrogen.    -   6) Derivatize dried extract with 100 μL of butanolic HCl for 30        min at 60° C.    -   7) Dry derivatized sample under heated air or nitrogen.    -   8) Reconstitute dried derivatized sample with reconstitution        solution for 10 minutes.    -   9) Cover plates with aluminum foil and place on autosampler.    -   10) Measure plates by MS/MS.

The second possibility, to measure enzyme activity, is generallyperformed by punching a small disc of a blood spot sample into amicrotiter well. Subsequently, a solution containing buffers is added toreconstitute an enzyme of interest. Once the enzyme is reconstituted, asecond solution containing a fluorescent analog to the natural substrateis added and the sample is incubated for a predetermined amount of timeto allow the reconstituted enzyme to act on the artificial substrate.Once sufficient time has elapsed, a third type of solvent is added tostop the reaction (precipitate.the enzyme). The plate containing thesample is then placed in a fluorometer and the fluorescence of theartificial product is measured. The fluorescence can then be correlatedto a particular level of enzyme activity.⁴⁻⁵

The advantages the MS/MS approach are that it allows for thesimultaneous analysis of several metabolite markers (acylcarnitines andamino acids) from a single sample and it alleviates environmentalfactors that affect enzyme activity such as exposure to light,temperature ranges, and humidity. The disadvantages are that the samplepreparation is lengthy and complicated as well as that it employsharmful solvents such as butanol in HCI that can be very deleterious topersonnel and equipment. Additionally and more importantly, is the factthat certain IEM cannot be screened for by MS/MS because the markermetabolites have many natural isomers also present in blood that preventthe measurement of their concentration. A case in point is galactosemetabolism in which the measurement of the marker galactose-1-phosphateis impeded because several other phosphorylated sugars present in bloodsuch as glucose-1-phospahate are isomeric with galactose-1-phosphate.Further, in many IEM, the levels of potential metabolite markers are toolow in blood to be measured effectively by MS/MS at a stage in which theaffected individual still does not show any symptoms. In these casesMS/MS would only be able to detect the marker metabolites at a clinicalstage that may be too late to administer effective preventive treatment.

Measuring enzyme activity by fluorometry alleviates some of thedisadvantages suffered by MS/MS. In case of isomeric interferences,there is no better discriminator of isomers than the natural enzymes.Enzymes have great specificity for their natural substrates such thatisomeric interferences are generally not an issue. Additionally, incases in which the levels of markers are too low to be measured directlyby MS/MS, one can reconstitute the enzyme of interest and add enoughartificial substrate to drive the reaction and be able to discriminatepatients with normal versus decreased enzyme activity. The disadvantagesof fluorometric methods are that one has to employ artificial substratesthat are tagged with a fluorescent group. Because of this, thesubstrates are different enough from their natural counter parts thatenzyme activity using such substrates is inherently reduced and thus thesensitivity of the assays. Additionally, because one estimates enzymeactivity by measuring fluorescence it is not practical to fully takeadvantage of multiplex analysis. In some cases, tags that wouldfluoresce at different wavelengths can be used, but instruments capableof reading multiple fluorescent labels can only handle a few of suchmarkers. Because of this, most enzyme activity assays employing sometype of light emission for measurement are single analyte assays.

There is need for improvements in the current methodologies used inclinical newborn screening laboratories. With the current technologies,these laboratories are forced to master several different technologiesto provide comprehensive testing. Additionally, as more and moreanalytes are added, the mainly single analyte-single test formatavailable requires that the patients donate more and more blood fortesting (a traumatic experience for the patient). Further, laboratoriesare forced to spend more and more time and resources when new analytesare added because the technologies available do not effectively allowfor multiplexing and expansion. Therefore, it is desirable to haveaccess to technology and methodology that can consolidate the twoclinical methods described above.

SUMMARY OF THE INVENTION

The present invention provides methods for simultaneous measurement ofenzyme activity and relatively polar and/or relatively non-polar analyteconcentration, thereby overcoming the deficiencies of prior artmethodologies. More specifically, the present invention does not requiresample derivatization or substrate tagging with fluorescent groups. Thepresent invention measurement of enzyme activity and metabolite elutionmay be performed in the same mixture, followed by tandem massspectrometry assay.

In general terms, the present invention is a method for geneticscreening of inborn errors of metabolism in a newborn by tandem massspectrometry, the method comprising the steps of: (a) obtaining a driedblood sample containing at least one enzyme, at least one relativelypolar metabolic analyte, and at least one relatively non-polar metabolicanalyte; (b) adding a solution of a relatively polar solvent to thedried blood sample so as to reconstitute the at least one enzyme in thedried blood sample and so as to extract the at least one relativelypolar analyte therefrom, thereby forming a first solution; (c)incubating the first solution with a substrate for the at least oneenzyme so as to create an enzymatic product; (d) adding a solution of arelatively non-polar solvent (this solution may contain acid at therequired concentration to precipitate the enzyme) to the first solutionso as to stop the reaction of the at least one enzyme and so as toextract the at least one relatively non-polar analyte, so as to form asecond solution; (e) measuring the second solution by tandem massspectrometry so as to assay the presence or concentration of the atleast one relatively polar analyte, the enzymatic product, and the atleast one relatively non-polar analyte.

The solution of a relatively polar solvent additionally may alsocomprise at least one component selected from the group consisting of:an internal standard for the at least one relatively polar analyte, aninternal standard(s) for the enzymatic product(s) to be measured, aninternal standard for the at least one relatively non-polar analyte, oneor more substrates for the enzyme(s), and a buffer. These internalstandard components may be labeled.

The solution of a relatively non-polar solvent may additionally compriseat least one component selected from the group consisting of: aninternal standard for at least one relatively polar analyte, an internalstandard(s) for the enzymatic product(s) to be measured, an internalstandard for at least one relatively non-polar analyte, and a buffer.These internal standard components may be labeled.

The present invention may be applied to a method for genetic screeningof inborn errors of metabolism in a newborn by tandem mass spectrometry.The method comprises placing a blood spot from the newborn into amicrotiter plate well. A solution of a polar solvent is then added tothe well so as to reconstitute enzymes in the blood spot and to extractpolar analytes, thereby forming a sample. The sample is then incubatedfor a predetermined period of time to extract metabolites and to allowsubstrate to product conversion by the reconstituted enzyme(s). A secondsolution is then added to the sample to stop enzymatic reactions. Atthis point the sample is mixed for a predetermined period of time(optional step) to allow additional metabolite extraction. The sample isthen measured by tandem mass spectrometry so as to identify analyteconcentration and enzyme activity. The analyte concentration and enzymeactivity are then used to determine whether the newborn is affected byany inborn errors of metabolism.

As used herein, the term “metabolite ” shall mean (1) a substanceproduced by metabolism; (2) a substance necessary for or taking part ina particular metabolic process; (3) a product of metabolism; (4) asubstance produced by metabolic action, as urea; or (5) any substanceinvolved in metabolism (either as a product of metabolism or asnecessary for metabolism).

As used herein the term “labeled ” shall include isotopic labeling,fluorescent labeling, and other forms of chemical labeling as are knownto those of ordinary skill in the art.

It is preferred that the solution of polar solvent comprises at leastone component selected from the group consisting of: an internalstandard for an analyte, an internal standard for an enzymatic productto be measured, a substrate, and a buffer. It is most preferred that atleast one of the at least one components are labeled.

The internal standard(s) may be one or more internal standards for eachclass of analyte, or even one internal standard per analyte. Theanalyte(s) may then be compared to the internal standard(s).

It is preferred that the second solution is less-polar than the solutionof polar solvent. It is preferred that the second solution comprises atleast one component selected from the group consisting of: an internalstandard for an analyte, an internal standard for an enzymatic productto be measured, and a buffer. It is most preferred that at least one ofthe at least one component is labeled.

The method may additionally comprise the step of adding at least onedetergent to the sample prior to mixing.

A second method for the genetic screening of inborn errors of metabolismin a newborn by tandem mass spectrometry of the present inventioncomprises placing a blood spot sample from the newborn into a microtiterplate well so as to form a sample. The microtiter plate well is treatedwith at least one pre-treater selected from the group consisting of: aninternal standard for the at least one relatively polar analyte, aninternal standard(s) for the enzymatic product(s) to be measured, aninternal standard for the at least one relatively non-polar analyte, oneor more substrates for the enzyme(s), and a buffer. These internalstandard components may be labeled. These pre-treaters are dry-coatedonto the microtiter well.

The method may additionally comprise the step of adding a polar solventto the well after placing the blood spot therein. Additionally, themethod may comprise adding a solution after incubation so as to stopenzymatic reactions. Optionally, the method may comprise mixing thesample for a predetermined period of time.

The sample (in the well) is incubated for a predetermined period of timebefore being measured by tandem mass spectrometry. Tandem massspectrometry is used to determine analyte concentration and enzymeactivity. The analyte concentration and enzyme activity data are used todetermine whether the newborn is affected by any inborn errors ofmetabolism.

Accordingly, the present invention includes both a combined metaboliteand enzyme activity sample preparation method, with a single tandem massspectrometry analysis in which the internal standards and substrates areincorporated within the solvents or are already present on themicrotiter well as dry-coated materials. The present inventionconsolidates the measurements of enzyme activity and metaboliteconcentration measurement into a single assay that does not requiresample derivatization. The present invention thus represents animprovement over current newborn screening methodologies that areperformed independently from each other.

Some of the many applications for the methods of the present inventioninclude but are not limited to assays for biotinidase deficiency, assayspertaining to disorders in carbohydrate metabolism (such asgalactosemia), assays for lyposomal storage disorders (such asmucopolysaccharidoses, sphingolipodoses, oligosaccharidoses andmucolipidoses), fatty acid β-oxidation and organic acid metabolicdisorders, amino acid metabolism disorders, and congenital adrenalhyperplasia. Other substances that may be determined from the methods ofthe present invention include but are not limited to carbohydrates, bileacids, very long chain fatty acids and steroids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a newborn screening method for metaboliteconcentration measurement by MS/MS in accordance with the prior art.

FIG. 2 is a schematic of a newborn screening method for enzyme activitymeasurement by fluorometry in accordance to prior art.

FIG. 3 is a schematic of a newborn screening method for combined enzymeactivity and metabolite concentration measurement by MS/MS in accordancewith one embodiment of the present invention.

FIG. 4 is a schematic of a newborn screening method for combined enzymeactivity and metabolite concentration measurement by MS/MS in accordancewith another embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

There is a need for improvement in the methodologies used in theclinical newborn screening laboratories. The present state of the artrequires laboratories to master several different technologies toprovide comprehensive testing. Additionally, as more and more analytesare added, the mainly single analyte-single test format available mayrequire that the patients donate more and more blood for testing (atraumatic experience for the patient). Further, laboratories are forcedto spend more and more time and resources when new analytes are addedbecause the technologies available do not effectively allow formultiplexing and expansion. Therefore, it is desirable to have access totechnology and methodology that can consolidate the two clinical methodsdescribed above. ⁶

With the current state of these technologies, it is not possible tocombine the two approaches. The main problem is that MS/MS methodsrequire sample derivatization. The conditions required to carry out thischemical modification of the sample may destroy both the enzymaticproducts as well as the enzymes; thus it may be impossible to measureenzyme activity with the current MS/MS methods. Additionally, many ofthe natural substrates and/or products involved in the metabolicpathways currently probed by enzyme activity assays belong to complexisomeric classes and/or are present in very low concentration or inconjugated forms. Therefore, it is not practical with the current MS/MSnewborn screening methodology to accurately measure their concentration.Transforming these enzyme activity assays into MS/MS metaboliteconcentration assays is thus currently impractical.

Previously, there has not existed a comprehensive methodology that couldperform both enzyme activity and analyte concentration measurementssimultaneously. However, the present invention provides a method thatdoes not require sample derivatization or substrate tagging withfluorescent groups and that consolidates the two types of assays into asingle sample preparation. The invention is a very simple and easy touse comprehensive clinical method.

First, a blood spot from a newborn is placed into microtiter plate wells(any plate format). To each well is added a solution of a polar solventcontaining internal standards for a multitude of analytes (whichever aredesired) and a multitude of substrates (whichever needed). In addition,this solution can contain internal standards for the enzymatic productsto be measured. This step accomplishes two tasks: a) enzymereconstitution and b) extraction of all polar analytes present in thesample. Buffers can also be added if necessary.

The internal standards are analogs of the analytes (preferably, but notrequired, isotopically labeled analogs). A multitude of standards can beadded (whichever are desired). The internal standards can also include(isotopically or other wise labeled) analogs of the enzymatic products.

The substrates (whichever needed) do not need to be tagged withfluorescent groups; they can be isotopically or other wise labeledanalogs to the natural substrate so that they have more physiologicaffinity to their corresponding enzyme. In this case, the labels aredifferent than in the enzymatic product internal standard to avoidconfusion.

The solution in this step is polar (for instance, any solvent varyingfrom pure water to any mixture of polar solvents and buffers). With thissolution we can reconstitute any enzyme and we can extract polarcompounds such as (not comprehensive list) amino acids, sugars,nucleobases, nucleotides, nucleosides, etc.

Next, the sample is incubated for a predetermined amount of time. Duringthis time, the reconstituted enzymes are acting on the specific addedsubstrates and the metabolites are being extracted simultaneously.

After the required incubation/extraction time is fulfilled, add anon-polar (or less polar than the “polar solution ” above) solvent toprecipitate the enzymes to stop the enzymatic reactions. At the sametime, this second solvent allows the extraction of non-polar to lesspolar analytes present in the sample. This solvent can also contain amultitude of non-polar (less polar) internal standards (includinginternal standards for the products of the enzymatic reactions). Forexample, if we add methanol at this step, enzymes will denature while atthe same time we can extract less polar analytes such as acylcarnitines,bile acids, fatty acids, less polar amino acids, organic acids, etc. Thesolution in this step is less polar than the polar solution previouslyadded. At some point (depending on polarity differences), the twosolutions may not be miscible with each other. In these instances,detergents can be added to homogenize the solutions.

It is preferred to mix the sample for a predetermined amount of time tohomogenize and extract the remaining analytes.

Next, the sample is measured by tandem mass spectrometry using isotopedilution MS/MS. Analyte concentration is measured by comparingintensities of the analytes against the intensities of the correspondinginternal standards of known concentration. Enzyme activity is measuredby determining the concentration of the labeled product. Theconcentration of the labeled product is measured by comparing theintensity of the product against the intensity of the correspondinginternal standard of known concentration. The concentration of theenzymatic product is then related to the incubation time to determineenzyme activity.

By this method one can measure both analyte concentrations and enzymeactivities for a multitude of analytes and enzymes out of a singlesample punch and sample preparation. With this method, one canpotentially screen from 25 to 50 IEM simultaneously.

It should be noted that all of the internal standards and substratesmentioned could already be coated on the well of the microtiter platesso that the solutions (i.e. the polar solution and theless-polar/non-polar solution) do not need to include them.

Further, the proportions of the polar and less-polar/non-polar solutioncan be adjusted to ensure proper extraction of each class of analytesand enzyme reconstitution. This will allow new analytes to be added tothe test menu with only minor changes to the method.

Additionally, this approach is flexible enough so that if one wants justto measure metabolite markers, one can consolidate the first tow ofsteps into a single one by making a solution of polar/less polarsolvents and in a single step extract both polar and non-polar analytes.We have found a mixture of 75/25 methanol/water (v/v) can extract bothpolar amino acids and less-polar acylcarnitines in one step and thatthey can be measured by MS/MS with no prior derivatization.

Alternatively, one can only measure enzyme activity if so desired byjust adding the corresponding substrates and internals standards(internal standards for the rest of the metabolites are not added).

In summary, the present invention includes a two-step protocol that doesnot require sample derivatization and that allows for the simultaneousmeasurement of metabolite concentrations and enzyme activities and thatallows for the simultaneous screening of a multitude of IEMs.

The method of the represent invention may also be practiced using thesample test containers and related methods described in co-pending U.S.patent application Ser. No. 09/474,604 by Ostrup, hereby incorporatedherein by reference.

FIG. 1 is a schematic of a newborn screening method for metaboliteconcentration measurement by MS/MS in accordance with the prior art.This method is used to extract and quantitate amino acids andacylcarnitines from newborn blood dried on filter paper. The amino acidsand acylcarnitines concentration profiles are used to screen fatty acidβ-oxidation, organic acid, and amino acid metabolic disorders innewborns (such as shown in U.S. Pat. Nos. 6,258,605 B1 and 6,455,321 B1to Chace; hereby incorporated herein by reference).

FIG. 2 is a schematic of a newborn screening method for enzyme activitymeasurement by fluorometry in accordance with the prior art. Thismethod, developed by Akie Fujimoto, et. al ⁷ is designed to screen forgalactosemia by reconstituting the enzyme galactose-1-phospateuridyltransferase (GALT) from newborn dried blood spots, adding afluorescent artificial substrate and monitor the rate of substrate toproduct conversion by fluorometry. The enzyme activity information isused to determine whether a newborn may be afflicted with galactosemia.

FIG. 3 shows a schematic of an assay procedure in accordance with oneembodiment of the invention. FIG. 3 shows that the approachesexemplified in FIGS. 1 and 2 are consolidated in the present inventionusing the following procedure: Dried blood spots (DBS) may be punchedinto one or more microtiter well(s). In the next step, the enzyme ofinterest and polar analytes (e.g. amino acids) are eluted with arelative polar solution (e.g. 100% water). This solution may contain allof the necessary internal standards and substrates. The mixture is thenincubated to allow enzyme activity and analyte extraction. In the nextstep, a relatively non-polar solvent (e.g., 100% MeOH) is added whichhalts the enzyme reaction and simultaneously extracts non-polar analytes(e.g. acylcarnitines). This second solution may contain all of thenecessary internal standards. Enzyme precipitation and analyteextraction are allowed to occur. The enzymatic product, polar analytesand non-polar analytes may then be analyzed/quantified by tandem massspectrometry. The analyte concentration and enzyme activity informationis then used to determine whether the newborn is afflicted with anyinborn error of metabolism.

FIG. 4 shows a schematic of an alternative assay procedure in accordancewith another embodiment of the invention. FIG. 4 shows that dried bloodspots (DBS) may be punched into one or more microtiter well(s). Thewells are already coated will all necessary internal standards andsubstrates. In the next step, the enzyme and the polar analytes (e.g.amino acids) are eluted with a relative polar solution (e.g. 100%water). The mixture is then incubated to allow enzyme activity. In thenext step, a relatively non-polar solvent (e.g., 100% MeOH) is addedwhich halts the enzyme reaction and simultaneously extracts non-polaranalytes (e.g. acylcarnitines). Enzyme precipitation and analyteextraction are allowed to occur. The enzymatic product, polar analytesand non-polar analytes may then be analyzed/quantified by tandem massspectrometry. The analyte concentration and enzyme activity informationis then used to determine whether the newborn is afflicted with anyinborn error of metabolism.

While the invention has been described in connection with what ispresently considered to be the most practical and preferred embodiments,it is to be understood that the invention is not to be limited to thedisclosed embodiment(s), but on the contrary, is intended to covervarious modifications and equivalent arrangements included within thespirit and scope of the appended claims, which are incorporated hereinby reference.

REFERENCES:

-   1. Rinaldo, P, and D. Matern. 2000. “Disorders of fatty acid    transport and mitochondrial oxidation: Challenges and dilemmas of    metabolic evaluation. Genetics in Medicine: 2000:2 (6):338-344.-   2. Rinaldo, P., D. Matern, and M. J. Bennett. 2002. Fatty Acid    Oxidation Disorders. Annu. Rev. Physiol. 64:477-502.-   3. McCaman, M W, and E. Robins. 1962. Fluorometric method for    determination of phenylalanine in serum. J. Lab. Clin. Med 59: 885.-   4. Chamoles, N. A., M. B. Blanco, D. Gaggioli, and C.    Casentini. 2001. Hurler-like Phenotype: Enzymatic Diagnosis in Dried    Blood Spots on Filter Paper. Clinical Chemistry 47(12):2098-2102.-   5. Chamoles, N. A., M. Blanco, and D. Gaggioli. 2001. Fabry disease:    diagnosis in dried blood spots on filter paper. Clinica Chimica    Acta. 308:195-196.-   6. Sweetman, L., 1996. “Newborn Screening by Tandem Mass    Spectrometry (MS-MS) Clinical Chemistry 42(3):345-346.-   7. Fujimoto, A., Y. Okano, T. Miyagi, G. Isshiki, and T. Oura. 2000.    Quantitative Beutler test for newborn mass screening of galactosemia    using a fluorometric microplate reader. Clinical Chemistry    46(6):806-810.

The foregoing references are hereby incorporated herein by reference.

1. A method for genetic screening of inborn errors of metabolism in anewborn by tandem mass spectrometry, said method comprising the stepsof: (a) obtaining a dried blood sample containing at least one enzyme,at least one relatively polar metabolic analyte, and at least onerelatively non-polar metabolic analyte; (b) adding a solution of arelatively polar solvent to said dried blood sample so as toreconstitute said at least one enzyme in said dried blood sample and soas to extract said at least one relatively polar analyte therefrom,thereby forming a first solution; (c) incubating said first solutionwith a substrate for said at least one enzyme so as to create anenzymatic product; (d) adding a solution of a relatively non-polarsolvent to said first solution so as to stop the reaction of said atleast one enzyme and so as to extract said at least one relativelynon-polar analyte, so as to form a second solution; and (e) measuringsaid second solution by tandem mass spectrometry so as to assay thepresence or concentration of said at least one relatively polar analyte,said enzymatic product, and said at least one relatively non-polaranalyte. (f)) using said analyte concentration and enzyme activity todetermine whether said newborn is affected by any inborn errors ofmetabolism
 2. The method according to claim 1 wherein said solution of arelatively polar solvent additionally comprises at least one componentselected from the group consisting of an internal standard for said atleast one relatively polar analyte, an internal standard for saidenzymatic product to be measured, a substrate for said at least oneenzyme, an internal standard for said at least one relatively non polaranalyte, and a buffer; or the method according to claim 1 wherein saidsolution of a relatively polar solvent additionally comprises at leastone component selected from the group consisting of a substrate for saidat least one enzyme and a buffer.
 3. The method according to claim 2wherein at least one of said internal standard for said at least onerelatively non-polar analyte, at least one of said internal standard forsaid at least one relatively polar analyte, at least one of saidinternal standard for said at least one enzymatic product to bemeasured, or a substrate for said at least one enzyme in said relativelypolar solvent is labeled.
 4. The method according to claim 1 whereinsaid solution of a relatively non-polar solvent additionally comprisesat least one component selected from the group consisting of: aninternal standard for said at least one relatively non-polar analyte, aninternal standard for said at least one substrate, and a buffer; or themethod according to claim 1 wherein said solution of a relativelynon-polar solvent additionally comprises at least one component selectedfrom the group consisting of an internal standard for said at least onerelatively polar analyte, an internal standard for said enzymaticproduct to be measured, an internal standard for said at least onerelatively non polar analyte, an internal standard for said at least onesubstrate, and a buffer.
 5. The method according to claim 4 wherein atleast one of said internal standard for said at least one relativelynon-polar analyte, at least one of said internal standard for said atleast one relatively polar analyte, at least one of said internalstandard for said at least one enzymatic product to be measured, atleast one of said internal standard for said at least one substrate insaid relatively non-polar solvent is labeled.
 6. The method according toclaim 1 additionally comprising the step of adding at least onedetergent to said sample prior to mixing said relatively polar andrelatively non-polar solutions.
 7. A method for genetic screening ofinborn errors of metabolism in a newborn by tandem mass spectrometry,said method comprising the steps of: (a) placing a blood spot samplefrom said newborn into a microtiter plate well so as to form a sample,said microtiter plate well treated with at least one pretreater; (b)incubating said sample for a predetermined period of time; (c) measuringsaid sample by tandem mass spectrometry so as to determine analyteconcentration and enzyme activity; and (d) using said analyteconcentration and enzyme activity to determine whether said newborn isaffected by any inborn errors of metabolism.
 8. The method according toclaim 7 additionally comprising the step of: adding a solution of arelatively polar solvent to said well so as to reconstitute said atleast one enzyme and so as to extract said at least one relatively polaranalyte therefrom, thereby forming a first solution.
 9. The methodaccording to claim 7 additionally comprising the step of: incubatingsaid first solution with a substrate for said at least one enzyme so asto create an enzymatic product.
 10. The method according to claim 7additionally comprising the step of: adding a solution of a relativelynon-polar solvent to said first solution so as to stop the reaction ofsaid at least one enzyme and so as to extract said at least onerelatively non-polar analyte, so as to form a second solution
 11. Themethod according to claim 7 additionally comprising the step of: mixingsaid second solution for a predetermined period of time.
 12. The methodaccording to claim 7 wherein said at least one pretreater is selectedfrom the group consisting of: an internal standard for a polar analyte,an internal standard for a non-polar analyte, an internal standard foran enzymatic product to be measured, a substrate, a detergent, and abuffer.
 13. The method according to claim 7 wherein at least one of saidat least one pretreater is labeled.